Nucleic acid protocols: Extraction and optimization

Graphical abstract


Introduction
Biomolecule extraction, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from a variety of starting biological materials to be used in downstream applications and other analytical or preparative purposes, is the most important first step in the molecular biology. The widely employed nucleic acid isolation methods can be divided into organic extraction method (phenol/chloroform), inorganic extraction method (salting out) and solid phase extraction method (solid matrix); moreover, four indispensable steps are generally required for successful nucleic acid purification: 1. Cell lysis through disruption of the cellular membranes, cyst wall or egg wall 2. Dehydration and precipitation of the cellular proteins (protein denaturation) 3. Separation of cellular proteins and other cellular components out of the nucleic acid

Precipitation and dissolving the nucleic acid
The routinely practised cell lysis step can be divided into three types to cope with different tissues, thereby achieving optimum nucleic acid yield: 1. Grinding in liquid nitrogen (mortar and pestle), such as different animal and plant tissues 2. Glass-bead grinding, for example, oocysts (e.g. Eimeria spp.), metacercariae (e.g. Fasciola spp.) and nematodes' eggs (e.g. eggs of Haemonchus contortus) 3. Repetitive pipetting, notable examples of it are animal cells and zoites of apicomplexan parasites, such as sporozoites, merozoites, tahyzoites and bradyzoites, and trypanosomal forms of Trypanosoma spp. and Leishmania spp., for example, trypomastigote, promastigote, amastigote and epimastigote.
In recent years, the development of molecular techniques has created a need for establishing simple and efficient novel methods of DNA and RNA extraction for PCR amplification and other related techniques. Carbohydrates, tannins, polyphenols and proteins in addition to hazardous organic solvents, such as phenol and chloroform are the major enemies of the embattled researchers. No existence for DNA or RNA extraction method that is suitable for all prokaryotic and eukaryotic organisms. Furthermore, there is an urgent need to address the insufficiency of reasonable environment for RNase to have DNA free of RNA and even for DNase to degrade the DNA.

Equipments
Mortar, Pestle, PCR machine, Microscope, Refrigerated Benchtop centrifuge (MIKRO200R, Germany), Weighing scale, Pipettes (20, 100, and 1000 ml), 15  2.4.1.1.1. Homogenization. 1 g of the liver was taken and cut into pieces then ground using a porcelain mortar and pestle in 3 ml of lysis buffer containing 900 ml of 10% SDS. The emulsion was transferred to micro-centrifuge tubes and 100 mg proteinase K was added per ml of emulsion solution, and incubated for 1 h at 50 C.
2.4.1.1.2. Phase separation. 350 ml of neutral saturated salt solution (NaCl) per ml was added to the previous emulsion, the microcentrifuge tube was capped and shaken gently by hand for 15 s, and then incubated at room temperature for 10 min. The microcentrifuge tube was centrifuged at 590 Â g for 15 min at room temperature with DNA remaining exclusively in the aqueous phase (see Fig. 1A for illustration).
2.4.1.1.3. DNA precipitation. The resulting aqueous phase was transferred into another micro-centrifuge tube, and mixed with two volumes of room temperature absolute ethyl alcohol. Then the micro-centrifuge tube was inverted several times for 10 s.
2.4.1.1.4. DNA wash. The supernatant was removed; the DNA pellet was washed once with 75% ethanol, and the DNA was precipitated out of the solution by centrifugation at 9500 Â g for 5 min.
2.4.1.1.5. DNA dissolving. The DNA pellet was allowed to dry for 5 min, and dissolved in DD water. Then the DNA was quantified and aliquoted to be stored at À20 C.
2.4.1.1.6. Removal of RNA from DNA preparation. 50 mg per ml RNase was added and the mixture was incubated for 1 h at 37 C.
Critical step: The treatment of DNA with RNase should be done in Tris buffer at the end of the extraction protocol. Salting out step can be repeated as before according to the protocol to obtain DNA with highest quality. The DNA can be precipitated and washed with 70% ethanol, and then the pellet can be dissolved in Tris-EDTA (TE) for DNA protection from degradation by metal dependent nucleases during storage.   (NaCl) was added into each tube of the previous emulsion mixture, and the micro-centrifuge tube was capped and gently shaken by hand for 15 s and then incubated at room temperature for 10 min. The micro-centrifuge tube was centrifuged at 590 Â g for 15 min at room temperature with RNA remaining exclusively in the aqueous phase (see Fig. 1B    unsporulated eimerian oocysts. As can be seen from Fig. 1D, the integrity of isolated total RNA was confirmed by 1.5% Agarose gel electrophoresis. 3.1.1.3. Assessing DNA and RNA for downstream applications. The ability to amplify a specific target from extracted DNA and RNA was proved using a pair of precise primers; MIC2-UP TATGGCTCGAGCGTTGTCGCTG and MIC2-D GTCAGGATGACTGTTGAGTGTC that were designed from the published Eimeria tenella microneme 2 (MIC2) mRNA sequence (ACCESSION FJ807654) (Fig. 1E). The primers were synthesized by AuGCT Biotechnology Synthesis Lab, Beijing, China. Fig. 1F Fig. 2A.

3.1.2.2.
Assessing DNA for downstream applications. The ability to amplify a specific target, such as 16S Ribosomal DNA from extracted DNA was proved using a pair of precise primers; forward GAPDH Primer, 5 0 -CAAGGTCATCCATGACAACTTTG-3 0 and the reverse GAPDH Primer, 5 0 -GTCCACCACCCTGTTGCTGTAG-3 0 that were provided with RevertAid TM First Strand cDNA Synthesis Kit to test the control sample (Fig. 2B).

DNA isolation
The average DNA purity ratio A 260 /A 280 was 1.87 AE 0.065 with DNA yield average of 48 AE 2.24 mg/1 ml (1 Â10 6 cells) of Escherichia coli bacterial cultures that were grown overnight in Luria Broth (LB) at 37 C. The integrity of isolated total DNA was confirmed by 1.5% Agarose gel electrophoresis as presented in Fig. 3A.

Assessing DNA for downstream applications
The ability to amplify a specific target, such as the E.coli 16S ribosomal RNA sequence from extracted DNA was proved using a pair of precise primers that were designed from the published E. coli 16S ribosomal RNA sequence (ACCESSION NO J01859/K02555/ M24828/M24833/M24834/M24835/M24836/M24837/M24911/ M24996) (Fig. 3B). The primers were synthesized by AuGCT Biotechnology Synthesis Lab, Beijing, China.

RNA isolation
The average purity of RNA samples was A 260 /A 280 ratio was 1.99 AE 0.01 and the quantity of RNA extracted from 1 Â10 6 E. coli was 22 AE 1.45 mg with integrity confirmed by 1.5% Agarose gel electrophoresis as can be seen from Fig. 3C. 3.3. Further assessment of RNA purity and integrity using trichostrongylid adult worm Total RNA from the barber's pole worm, Haemonchus contortus was isolated employing salting out (acidic condition) followed by DNase digestion. For contaminant detection, the A 260 /A 280 and A 260 /A 280 values (i.e. for detection of protein contaminants and residual chemical contamination, such as EDTA and SDS) were 2 AE 0.02 and 2.076 AE 0.024, respectively [1][2][3]. Additionally, Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA) was used in conjunction with the traditional 1.5% agarose gels for RNA integrity assessment (Fig. 4,Table 1).
These results present a simplified, semi-unified, effective, and toxic material free protocol for extracting DNA and RNA from different prokaryotic and eukaryotic sources exploiting the physical properties of the negatively charged molecules; DNA and RNA. The positively ions of saturated salt solution neutralize the negatively charged phosphate groups of the DNA and RNA backbone. Furthermore, in neutral saturated salt conditions, DNA will remain in the aqueous layer. However, RNA will partition into the aqueous layer by carrying out acidic saturated salt solution extraction.
Yield and quality are the ultimate goal for any researchers during DNA extraction procedure. Doubtless, the quality increases by getting RNA free of DNA contamination. Previous published studies failed to resolve this issue [4][5][6][7][8][9][10][11][12] The most common protocols used the chelating agent, ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS) as a detergent, and sodium chloride as a stabilizer in the lysis buffer. The high affinity of EDTA to divalent cations, such as Ca 2+ , Mn 2+ and Mg 2+ , which act as cofactors for nucleases could inhibit the degradation of DNA and RNA by DNases and RNases respectively [13][14][15]. SDS is an anionic detergent for cell and nucleus lysis to release ribonucleic and deoxyribonucleic acids. The nucleases; ribonuclease (RNase) and deoxyribonuclease (DNase) activities were inhibited by SDS [16,17]. The electrostatic repulsion between the two negatively charged helix strands destabilizing the helix was counteracted by positively charged sodium chloride [18]. The shielding effect of monovalent sodium cations leads to DNA and RNA stabilization through neutralization of the negative charge on the sugar phosphate backbone as is demonstrated in Fig. 5 [19]. Elevated salt concentration, SDS and EDTA were used to inhibit nuclease activity during extraction of DNA from tissues or organisms with high nuclease activity [20]. The use of sodium chloride in the lysis buffer decreases the susceptibility of DNA and RNA to be attacked by the action of nucleases possibly due to steric hindrance. Additionally, salting out denatures proteins and leaves nucleic acids intact. This is the most potent way of expeditiously inactivating nucleases.
We pointed out that DNA and RNA are under triple protection (i.e. EDTA, SDS and NaCl) and this environment is unsuitable for RNase to get DNA free of RNA and even for DNase to degrade DNA. Our conclusion is supported by results from treatments of different prokaryotic and eukaryotic sources as illustrated in Figs. 6A-C. The complete removal of RNA under the effect of RNase is achieved when RNase is eventually added (i.e. in Tris buffer without EDTA), which gives optimal quality with any DNA extraction protocols.
The polar phosphate groups of DNA and RNA can electrostatically interact with the polar environment allowing them to be easily dissolved in Tris buffer; therefore, the treatment of DNA and RNA with RNase and DNase respectively should be done in Tris buffer at the end of the extraction protocol as a standard measure. Salting out step can be repeated as before according to the protocol to obtain DNA with the highest quality without major changes in the nucleic acid yield. The DNA can be precipitated and washed with ethanol, and then the pellet can be dissolved in Tris-EDTA (TE) for DNA and RNA protection from degradation by divalent-metaldependent nucleases during storage.

Conclusion
A simplified, semi-unified, effective, and toxic material free protocol for extracting DNA and RNA utilizing the physicochemical properties of nucleic acids has been described. Moreover, the unsuitable environment for endonucleases, such as RNase and DNase to have DNA liberated of RNA and even for DNase to degrade the DNA respectively has been addressed, and an appropriate alternative protocol has been presented that could be the top-first in the field of molecular biology.